- Email: [email protected]
Quantitative determination of human fibrinogen in plasma following carbamylation by laurell electrophoresis in antibody containing gel
281
CLINICA CHIMICA ACTA
CC* 4448
QUANTITATIVE FOLLOWING ANTIBODY
H. GRY
DETERMINATION CARBAMYLATION
CONTAINING
NIELSEN
OF HUMAN BY
LAURELL
FIBRINOGEN
IN PLASMA
ELECTROPHORESIS
IN
GEL
AND B. WEEKE
Medi-Lab
a.~., Copenhagen
(Received
January
and The
University
Pyoteix
Laboratory,
Co_@nhagen
(Dmnmvk)
18, 1971)
SUMMARY
Anodic precipitates of fibrinogen suitable for quantitation can be obtained by Laurel1 electrophoresis in antibody containing gel at pH 8.6 after carbamylation. The results obtained are in agreement with the fibrin estimation by means of a Biuret method. With a routine procedure the mass concentrations of plasma fibrinogen within the range 1.4-11.0 g/l can be estimated with a coefficient of variation of 5.4%. In 45 normal subjects the mean concentration of plasma fibrinogen was 3.6 g/l and the 95 per cent normal range 2.2-4.9
g/l.
INTRODUCTION
The most frequently used methods for determination of fibrinogen are based on the estimation of the fibrin precipitate adding calcium or thrombin to plasmal. By Laurel1 electrophoresis in antibody containing agarosez at pH 8.6, the fibrinogen will form cathodic-anodic protein precipitates with broad diffusion rings round the applications. Thus the height of the precipitates is not a good measure of the fibrinogen concentration. As previously demonstrated, the electrophoretic mobility of many proteins can be altered by carbamylation using a reaction with potassium cyanate3. The electrophoretic mobility of fibrinogen is hereby changed from beta- to alpha-mobility. In the present study it is demonstrated that carbamylated human fibrinogen by electrophoresis in antifibrinogen containing agarose yields anodic precipitates well suited for quantitative determination of plasma fibrinogen. The results were compared with determinations by means of a Biuret method. METHODS
Laurel1 electrophovesis in antibody coxtaining gel2 The electrophoresis is carried out by 2.5-3 V/cm overnight
Clin. Chim.
at 15'.The compoActa,
35 (1971)
281-284
282
NIELSEN, WEEKE
sition of the gel is: I g agarose (L’Industrie Biologique Francaise), 1.3 ml rabbit antihuman fibrinogen (Dakopatts), IOO ml barbitone buffer (pH 8.6, ionic strength 0.02). Citrated @asma is prepared by bleeding 4.5 ml blood into tubes containing 0.5 ml sodium citrate (0.1 nil). Due to this dilution of the plasma and using a mean hematocrit value of 44 the results were multiplied by 1.2 in order to give the concentration in g/l plasma. Freeze-dried fibrinogen from KABI is used as reference for both methods; it is dissolved in distilled water divided into portions corresponding to daily requirements, and stored at -18”. The concentration of protein was adjusted to be 10.0 g/l as determined by the N-analysis performed according to Kjeldahl, and corrected for a small amount of IgG (3y0 relative). Carbawylation. Equal volumes of titrated plasma or reference solution and freshly prepared potassium cyanate 2 PI react at 37’ for 30 minutes, and are then placed in cold water (below 12’). Dilutions. For establishing the reference curve the carbamylated reference solution is diluted 241-121-61-31 fold (fibrinogen concentrations: 22 mg/l, 62 mg/l, 122 mg/l, 162 mg/l). The carbamylated
plasma is diluted 31 fold.
Biuret method1 1500 ~1 0.9 per cent sodium chloride and 1500 ~1 thrombin solution (IOO NIH U/ml, Hoffmann-La Roche & Co.) are added to 300 ~1 titrated plasma or reference solution. The mixture is left for 30 minutes at 37’. The resulting clot is isolated, dried, and redissolved in 3000 ~1 of Biuret reagent at 37O for 2 hours. The optical density is read at 546 nm. RESULTS AND DISCUSSION Fig. I (holes 1-4 and 9-12) demonstrates
that fibrinogen
yields cathodic-anodic
precipitates with broad diffusion rings round the holes of application in Laurell’s electrophoresis. After carbamylation (holes 5-8) the carbamylated fibrinogen yields anodic precipitates only. The relation between the height of the precipitates and the concentration of fibrinogen shows straight to slightly arched curves. Therefore the above-mentioned four dilutions of the reference are used in the daily routine for establishing the reference curve. By diluting carbamylated plasma 31 fold the fibrinogen concentrations within the range 1.4-11.0 g/l may be interpolated on the curve. Due to the concentration of cyanate the carbamylated samples should be diluted at least 3 fold. Otherwise a reaction with the antibodies in the gel around the holes will occur. By this dilution concentrations of fibrinogen as low as 0.14 g/l can be determined and by extrapolation still smaller amounts can be detected. In the present study the electrophoresis has been carried out overnight at a low voltage. It is, however, possible to finish the electrophoresis in z hours using a higher voltage (about IO V/cm) and adding more antifibrinogen to the gel (about 50%). In order to compare the purified fibrinogen in the reference with the genuine fibrinogen in plasma, known amounts of the reference were added to plasma samples with known concentrations of fibrinogen. Recoveries of 97-105% were found, indiC2in. Chinz.
Acta,
35 (1971) z81--28z+
~~TE~MI~ATION
OF PLASMA
FIBRINOGEN
283
eating that the fibrinogen in the plasma and the reference is immunologically comparable. This comparisan together with the N-analysis according to Kjeldahl are carried out on each new batch of fibrinogen reference.
Fig. I. Plasma fibrinogen estimated by Laurel1 electrophoresis in antifibrinogen containing gel. The diameter of the holes is 4 mm. Holes 1-4 contain 22 mg/l, 62 mg/l, 122 mg/l, and 162 mg/l of noncarbamylated fibrinogen, holes 5-X the same amounts of carbamylated fibrinogen, and holes g-12 IO times the amounts of non-carbamyiated fibrinogen,
Fibrinogen by the Biuret
method ingfi
Fig. 2. The correlation between the concentration of fibrinogen in 25 different plasma samples estimated by the Biuret method and immunochemically by Laurel1 electrophoresis. C&z. Chim. Acta, 35 (1971) 281-284
284
NIELSEN,
WEEKE
15 double estimations of a plasma sample were carried out on plates with three different commercial evailable antisera and the same reference. The mean values of the fibrinogen concentrations were respectively 3.98 g/l. 3.95 g/l, and 3.97 g/l with an “intraplate” relative standards deviation of 3.0. Different antisera did not influence the determination of fibrinogen. In a plasma sample with 3.3 g/l which has been determined on x7 different days during 2 months a standard deviation of 0.18 g/l (relative standard deviation of 5.4) was found. On different plasma samples determinations of fibrinogen have been carried out by means of Laurell’s electrophoresis in antibody containing gel and by a Biuret method. The correlation is significant (P < 0.001) and according to the equation y = 1.19~ -0.27 g/l (1.93 < x < 10.40, n = 25, y = 0.98, P < 0.001) where y is fibrinogen by the immunocllenlical method and x fibrinogen by a Biuret method, the fibrinogen concentrations determined immunochemically give slightly higher values than a Biuret method, but the difference was not significant (P > 0.05) (Fig. 2). Storage at 4°C for I week did not cause any significant change in the fibrinogen concentration, while storage for z weeks caused an average loss of about 20%. Samples were stored at -18” for 3 months without any apparent change. The fibrinogen concentrations in 45 healthy subjects aged 19-50 years are shown in Table I. TARLE
I AND 95T0 RASGE
MEAX YEARS
FOR THE CONCENTRATION
OF
FIBRINOGEN
IN
4j
R’ORMAL
SUBJECTS,
19.-jO
OLD
n1a1es
-
Females off oral contraceptives 12emales on oral contraceptives All persons Observed ran*@ 5.1). = standard
Kumbev
Mema g/l
15
3.2
1.7-4.7
15 r5 45
3.6 3.9 3-6
2.7-4.5 2.6-5.2 2.1-4.9 2.4-6.1
Xean
-i_ z S.D. g/l
deviation
The values were normally distributed. The slightIy lower concentrations in males are not significant (P > 0.05). The difference between the 15 females off and on oral contraceptives (treatment 1/z-4 years) is not significant, either (P > 0.05). So a common normal range for the 3 groups is used. No change in the fibrinogen concentration with age could be demonstrated. REFERENCES I R. RICHTERICH, in R. RICHTERICH (Ed.), Klinische Chemie, S. Karger, 2 C.-B. LAURELL, Ann. Biochenz., 15 (1966) 45. 3 R. ?VHERE, Scmd. J. Cl&. Lab. Invest., 21 (1968) 351. c&t.
Chim.
Acta,
35 (1971)
2111-28~
Basel/Ncw
York,
1965.
CLINICA CHIMICA ACTA
CC* 4448
QUANTITATIVE FOLLOWING ANTIBODY
H. GRY
DETERMINATION CARBAMYLATION
CONTAINING
NIELSEN
OF HUMAN BY
LAURELL
FIBRINOGEN
IN PLASMA
ELECTROPHORESIS
IN
GEL
AND B. WEEKE
Medi-Lab
a.~., Copenhagen
(Received
January
and The
University
Pyoteix
Laboratory,
Co_@nhagen
(Dmnmvk)
18, 1971)
SUMMARY
Anodic precipitates of fibrinogen suitable for quantitation can be obtained by Laurel1 electrophoresis in antibody containing gel at pH 8.6 after carbamylation. The results obtained are in agreement with the fibrin estimation by means of a Biuret method. With a routine procedure the mass concentrations of plasma fibrinogen within the range 1.4-11.0 g/l can be estimated with a coefficient of variation of 5.4%. In 45 normal subjects the mean concentration of plasma fibrinogen was 3.6 g/l and the 95 per cent normal range 2.2-4.9
g/l.
INTRODUCTION
The most frequently used methods for determination of fibrinogen are based on the estimation of the fibrin precipitate adding calcium or thrombin to plasmal. By Laurel1 electrophoresis in antibody containing agarosez at pH 8.6, the fibrinogen will form cathodic-anodic protein precipitates with broad diffusion rings round the applications. Thus the height of the precipitates is not a good measure of the fibrinogen concentration. As previously demonstrated, the electrophoretic mobility of many proteins can be altered by carbamylation using a reaction with potassium cyanate3. The electrophoretic mobility of fibrinogen is hereby changed from beta- to alpha-mobility. In the present study it is demonstrated that carbamylated human fibrinogen by electrophoresis in antifibrinogen containing agarose yields anodic precipitates well suited for quantitative determination of plasma fibrinogen. The results were compared with determinations by means of a Biuret method. METHODS
Laurel1 electrophovesis in antibody coxtaining gel2 The electrophoresis is carried out by 2.5-3 V/cm overnight
Clin. Chim.
at 15'.The compoActa,
35 (1971)
281-284
282
NIELSEN, WEEKE
sition of the gel is: I g agarose (L’Industrie Biologique Francaise), 1.3 ml rabbit antihuman fibrinogen (Dakopatts), IOO ml barbitone buffer (pH 8.6, ionic strength 0.02). Citrated @asma is prepared by bleeding 4.5 ml blood into tubes containing 0.5 ml sodium citrate (0.1 nil). Due to this dilution of the plasma and using a mean hematocrit value of 44 the results were multiplied by 1.2 in order to give the concentration in g/l plasma. Freeze-dried fibrinogen from KABI is used as reference for both methods; it is dissolved in distilled water divided into portions corresponding to daily requirements, and stored at -18”. The concentration of protein was adjusted to be 10.0 g/l as determined by the N-analysis performed according to Kjeldahl, and corrected for a small amount of IgG (3y0 relative). Carbawylation. Equal volumes of titrated plasma or reference solution and freshly prepared potassium cyanate 2 PI react at 37’ for 30 minutes, and are then placed in cold water (below 12’). Dilutions. For establishing the reference curve the carbamylated reference solution is diluted 241-121-61-31 fold (fibrinogen concentrations: 22 mg/l, 62 mg/l, 122 mg/l, 162 mg/l). The carbamylated
plasma is diluted 31 fold.
Biuret method1 1500 ~1 0.9 per cent sodium chloride and 1500 ~1 thrombin solution (IOO NIH U/ml, Hoffmann-La Roche & Co.) are added to 300 ~1 titrated plasma or reference solution. The mixture is left for 30 minutes at 37’. The resulting clot is isolated, dried, and redissolved in 3000 ~1 of Biuret reagent at 37O for 2 hours. The optical density is read at 546 nm. RESULTS AND DISCUSSION Fig. I (holes 1-4 and 9-12) demonstrates
that fibrinogen
yields cathodic-anodic
precipitates with broad diffusion rings round the holes of application in Laurell’s electrophoresis. After carbamylation (holes 5-8) the carbamylated fibrinogen yields anodic precipitates only. The relation between the height of the precipitates and the concentration of fibrinogen shows straight to slightly arched curves. Therefore the above-mentioned four dilutions of the reference are used in the daily routine for establishing the reference curve. By diluting carbamylated plasma 31 fold the fibrinogen concentrations within the range 1.4-11.0 g/l may be interpolated on the curve. Due to the concentration of cyanate the carbamylated samples should be diluted at least 3 fold. Otherwise a reaction with the antibodies in the gel around the holes will occur. By this dilution concentrations of fibrinogen as low as 0.14 g/l can be determined and by extrapolation still smaller amounts can be detected. In the present study the electrophoresis has been carried out overnight at a low voltage. It is, however, possible to finish the electrophoresis in z hours using a higher voltage (about IO V/cm) and adding more antifibrinogen to the gel (about 50%). In order to compare the purified fibrinogen in the reference with the genuine fibrinogen in plasma, known amounts of the reference were added to plasma samples with known concentrations of fibrinogen. Recoveries of 97-105% were found, indiC2in. Chinz.
Acta,
35 (1971) z81--28z+
~~TE~MI~ATION
OF PLASMA
FIBRINOGEN
283
eating that the fibrinogen in the plasma and the reference is immunologically comparable. This comparisan together with the N-analysis according to Kjeldahl are carried out on each new batch of fibrinogen reference.
Fig. I. Plasma fibrinogen estimated by Laurel1 electrophoresis in antifibrinogen containing gel. The diameter of the holes is 4 mm. Holes 1-4 contain 22 mg/l, 62 mg/l, 122 mg/l, and 162 mg/l of noncarbamylated fibrinogen, holes 5-X the same amounts of carbamylated fibrinogen, and holes g-12 IO times the amounts of non-carbamyiated fibrinogen,
Fibrinogen by the Biuret
method ingfi
Fig. 2. The correlation between the concentration of fibrinogen in 25 different plasma samples estimated by the Biuret method and immunochemically by Laurel1 electrophoresis. C&z. Chim. Acta, 35 (1971) 281-284
284
NIELSEN,
WEEKE
15 double estimations of a plasma sample were carried out on plates with three different commercial evailable antisera and the same reference. The mean values of the fibrinogen concentrations were respectively 3.98 g/l. 3.95 g/l, and 3.97 g/l with an “intraplate” relative standards deviation of 3.0. Different antisera did not influence the determination of fibrinogen. In a plasma sample with 3.3 g/l which has been determined on x7 different days during 2 months a standard deviation of 0.18 g/l (relative standard deviation of 5.4) was found. On different plasma samples determinations of fibrinogen have been carried out by means of Laurell’s electrophoresis in antibody containing gel and by a Biuret method. The correlation is significant (P < 0.001) and according to the equation y = 1.19~ -0.27 g/l (1.93 < x < 10.40, n = 25, y = 0.98, P < 0.001) where y is fibrinogen by the immunocllenlical method and x fibrinogen by a Biuret method, the fibrinogen concentrations determined immunochemically give slightly higher values than a Biuret method, but the difference was not significant (P > 0.05) (Fig. 2). Storage at 4°C for I week did not cause any significant change in the fibrinogen concentration, while storage for z weeks caused an average loss of about 20%. Samples were stored at -18” for 3 months without any apparent change. The fibrinogen concentrations in 45 healthy subjects aged 19-50 years are shown in Table I. TARLE
I AND 95T0 RASGE
MEAX YEARS
FOR THE CONCENTRATION
OF
FIBRINOGEN
IN
4j
R’ORMAL
SUBJECTS,
19.-jO
OLD
n1a1es
-
Females off oral contraceptives 12emales on oral contraceptives All persons Observed ran*@ 5.1). = standard
Kumbev
Mema g/l
15
3.2
1.7-4.7
15 r5 45
3.6 3.9 3-6
2.7-4.5 2.6-5.2 2.1-4.9 2.4-6.1
Xean
-i_ z S.D. g/l
deviation
The values were normally distributed. The slightIy lower concentrations in males are not significant (P > 0.05). The difference between the 15 females off and on oral contraceptives (treatment 1/z-4 years) is not significant, either (P > 0.05). So a common normal range for the 3 groups is used. No change in the fibrinogen concentration with age could be demonstrated. REFERENCES I R. RICHTERICH, in R. RICHTERICH (Ed.), Klinische Chemie, S. Karger, 2 C.-B. LAURELL, Ann. Biochenz., 15 (1966) 45. 3 R. ?VHERE, Scmd. J. Cl&. Lab. Invest., 21 (1968) 351. c&t.
Chim.
Acta,
35 (1971)
2111-28~
Basel/Ncw
York,
1965.